Marburg viruses (MARV), which cause outbreaks of viral hemorrhagic fever in humans with mortality rates of up to 90%, are category A priority pathogens. However, the discovery of filovirus antivirals and molecular probes has been hampered by the need for biosafety level (BSL) 4 containment for live virus studies. An alternative to the use of live virus for such studies is to develop assays that recapitulate specifc host-viral interactions that are required for pathogenesis. These interactions can be characterized and targeted for therapeutic development without the need for live virus, alleviating the need for BSL4 containment. Additionally, such screens will be well-positioned to take advantage of sophisticated HTS infrastructure that would enable antifiloviral identification a many non-BSL4 facilities. This proposal focuses on a unique aspect of MARV biology that was recently experimentally characterized by the participating groups. We showed that an interaction between MARV VP24 (mVP24) and Kelch-like ECH-associated protein 1 (Keap1) results in activation of cytoprotective antioxidant responses through the stabilization/nuclear localization o transcription factor Nuclear factor (erythroid-derived 2)-like 2 (Nrf2). In the nucleus, Nrf2 binds antioxidant response element (ARE) sequences to promote transcription of a cytoprotective gene expression program. Keap1 is a specificity factor for Cul3 ubiquitin ligase that regulates the stability and the level of Nrf2 in cells. We demonstrated that the Keap1 Kelch domain binds mVP24 with high affinity, freeing Nrf2 to activate gene expression. Biophysical and modeling studies indicate that mVP24 interacts with Keap1 in a manner that is unique and distinct from the binding mode used by other Keap1 binders, including Nrf. We hypothesize that induction of the cytoprotective pathways will sustain the viability of infected cells and facilitate virus replication. Thus, targeting this novel aspect of MARV replication may provide insight into viral replication and opportunities for countermeasure development. The goals of this proposal are to develop optimized primary and secondary screening assays, based on ARE reporter genes and on fluorescence polarization approaches, to identify small molecules that prevent mVP24-mediated activation of an ARE response, and to perform a small-scale pilot screen that allows us to establish criteria for hit selection. These efforts will pave the way for larger, full-scale mall molecule screens with the potential to identify novel anti-Marburg virus compounds that inhibit mVP24 activation of an ARE response, including molecules that disrupt mVP24-Keap1 interaction.